Dissertations / Theses on the topic 'Therapeutic enzymes'

Cette thèse concerne le développement d’outils microfluidique pour l’ingénierie d’enzymes d’intérêt thérapeutique. La microfluidique à base de gouttelettes présente un énorme potentiel dans le domaine de la biologie quantitative. Nous développons des outils microfluidiques pour l’évolution dirigée de l’enzyme L-asparaginase, enzyme utilisée comme traitement de laleucémie lymphoblastique aiguë. Ce traitement est basée sur une enzyme d’origine bactérienne,ce qui conduit à déclencher des réactions immunitaires qui se traduit par l’interruption du traitement, souvent fatale pour le patient. Cependant, une version humaine de l’enzyme L-asparaginase, qui est moins immunogénique, n’est à l’heure actuelle pas suffisamment active pour être utilisée. L’objectif principal de cette thèse est d’alors d’analyser et de cribler des banques de mutants d’enzymes en utilisant des méthodes classiques de mutagenèse et d’analyser chaque mutant individuellement par le biais de la microfluidique. Pour cela, plusieurs systèmes microfluidiques ont été développés et optimisés afin de répondre à différents critères de sélection pour l’analyse et la sélection de l’enzyme L-asparaginase. La version bactérienne a servi de contrôle positif pour l’optimisation des systèmes microfluidiques afin de pouvoir analyser et de cribler des banques de mutants de la version humaine de l’enzyme L-asparaginase<br>This thesis deals with the development of microfluidic tools for the engineering ofenzymes of therapeutic interest. Droplet microfluidics has enormous potential in the field ofquantitative biology. We are developing microfluidic tools based on the directed evolutionof the enzyme L-asparaginase, an enzyme used to treat acute lymphoblastic leukemia. Thistreatment is based on an enzyme of bacterial origin, which leads to immune reactions thatresult in the interruption of treatment, often fatal for the patient. However, a human version ofthe enzyme L-asparaginase, which is less immunogenic, is currently not sufficiently active to beused. The main objective of this thesis is to analyze and screen enzyme mutant libraries usingstandard mutagenesis methods and to analyze each mutant individually through microfluidics.For this, several microfluidic systems have been developed and optimized for different selectioncriteria for the analysis and selection of the enzyme L-asparaginase. The bacterial versionserving as a positive control for the optimization of microfluidic workflows to analyze andscreen mutant libraries of the human version of the enzyme L-asparaginase

The \(Escherichia\) \(coli\) enzyme nitroreductase has been proposed as a candidate for the Gene-Directed Enzyme Prodrug Therapy approach in treating cancer. Structural studies on the enzyme were instigated in a first step towards improving enzyme activity. The enzyme was crystallized with the substrate analogue, nicotinic acid, and the structures of three crystal forms obtained. The fold has a mixed a/P structure, with a molecule of nicotinic acid bound next to the FMN cofactor. Several active site residues were identified as candidates for mutation. This procedure produced many mutant enzymes with increased catalytic activity. One double and four single mutants were chosen from these and crystal structures determined. The resulting information from this, and the establishment of a proof of principle, provides the basis for iterative cycles of enzyme improvement. The human hydroxysteroid dehydrogenase AKR1C3 has been proposed to play a role in prostaglandin metabolism. Its inhibition by non-steroidal anti-inflammatory drugs may be important in a tumour differentiation strategy. AKR1C3 was crystallized, and the structure solved with bound nucleotide cofactor and several inhibitors, including the drugs indomethacin and flufenamic acid. Having obtained information on drug binding to AKR1C3, selective inhibitors can be designed, avoiding inhibition of "housekeeping" enzymes such as cyclo-oxygenases.

The focus of disease research often surrounds therapeutic pathway identification and the subsequent investigation of proteins or compounds that potentially interfere with disease mechanisms. However, finding targets and effective therapeutic domains often overshadows another important aspect of drug delivery and efficacy; the method of domain conjugation. Unfortunately the combination of good therapeutic components and good therapeutic design can often be amiss, with differing skills and groups needed to marry the two together. In recognition of this, there are new techniques emerging that aim to not only address old problems of stable conjugation, but incorporating new knowledge of drug design. This research takes a reverse approach to drug design in order to better comprehend requirements which are often an afterthought; first looking at conjugation technique and then how it can best be exploited. Thereby, exploring how different conjugation methods can be used to complement existing therapeutic and internalisation strategies. Here, two methods of conjugation were investigated through attachment of bacterial toxin domains derived from botulinum A and diphtheria, with the intention of targeting neuroblastoma cells using a variety of specific and non-specific pathways. Both conjugation techniques proved to be stable in vitro and capable of binding a variety of domains consistently. The data here also emphasized the importance of endosomal escape proteins to increase the translocation of catalytic domains into the cytosol. It was demonstrated that the conjugates formed were able to facilitate the binding between targeting and catalytic domains, enabling specific neuroblastoma internalisation. Moreover, that three domains were able to conjugate together using a linking method and could form a co-operative triple-functioning complex.

Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutique<br>Phospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach

Les leishmanioses sont des maladies tropicales négligées provoquées par un protozoaire parasite du genre Leishmania, et transmises par un insecte vecteur, le phlébotome. Les leishmanioses menacent 310 millions de personnes dans 98 pays à travers le monde. Les traitements antileishmaniens actuels sont limités et présentent des problèmes majeurs de toxicité et d'émergence de chimiorésistance. Dans ce contexte, il est nécessaire de développer de nouveaux agents antileishmaniens spécifiquement dirigés contre une cible thérapeutique chez le parasite. La GDP-Mannose Pyrophosphorylase (GDP-MP) est une cible thérapeutique essentielle à la survie du parasite à la fois in vitro et in vivo. Plusieurs différences ont été identifiées dans le site actif de GDP-MPs leishmaniennes par rapport à l'enzyme humaine, montrant ainsi l'intérêt de cette cible thérapeutique dans le développement de nouveaux traitements contre la leishmaniose. La GDP-MP catalyse la synthèse du GDP-mannose, la forme activée du mannose, brique moléculaire importante dans les processus de glycosylation et la synthèse de glycoconjugués essentiels à la reconnaissance hôte-parasite. Ce travail de thèse a consisté à produire et purifier les GDP-MPs de 3 espèces de parasites (Leishmania infantum, Leishmania donovani et Leishmania mexicana) ainsi que l'homologue humaine dans le but de comparer leurs propriétés enzymatiques. A partir d'inhibiteurs potentiels conçus et synthétisés sur la base de modèles moléculaires de GDP-MPs leishmaniennes et humaine, 100 composés ont été évalués sur les enzymes purifiées et sur les parasites in vitro. Cette analyse nous a permis de sélectionner des composés spécifiquement dirigés contre la cible du parasite et présentant une activité antileishmanienne. Nous avons également initié une étude de l'expression et de la localisation de la GDP-MP après traitement par les composés les plus intéressants. Ces composés pourront par la suite être utilisés comme outils pharmacologiques pour le développement de nouveaux agents antileishmaniens spécifiques<br>Leishmaniases are Neglected Tropical Disease (NTD)caused by a protozoan parasite of the genus Leishmania and transmitted by an insect vector, the phlebotomine sandfly. Leishmaniases threaten 310 millions people in 98 countries around the world. Current antileishmanial treatments are limited and present major issues of toxicity and drug resistance emergence. In this context, it is necessary to develop new specific antileishmanial drugs specifically directed against a therapeutic target in the parasite.The GDP-Mannose Pyrophosphorylase (GDP-MP) is a therapeutic target which has been described to be essential for parasite survival both in vitro and in vivo. Several differences have been identified in the active site of leishmanial GDP-MPs compared to the human counterpart, showing the prominence of this therapeutic target in the development of new treatments against leishmaniasis. The GDP-MP catalyzes the synthesis of GDP-Mannose,the activated form of mannose, an important molecular constituent of the glycosylation processes involved in the biosynthesis of glycoconjugates which are essential for host-parasite recognition. My thesis work consisted in the production and purification of GDP-MPs from 3 Leishmania species (Leishmania donovani,Leishmania infantum and Leishmania mexicana)and from humanin order to compare their enzymatic properties. From potential inhibitors designed and synthesized on the basis of leishmanial and human GDP-MP molecular models, 100 compounds were evaluated on purified enzymes and on parasites in vitro. These analyses allowed us to select some compounds which are specifically directed against the parasite target and presenting antileishmanial activities. We have also initiated a study of expression and localization of GDP-MP after treatment with the most potent compounds. These compounds will be used as pharmacological tools for the development of new specific antileishmanial drugs

Lysosomal storage disorders (LSD) are a large group of inherited genetic diseases caused by impaired activity of lysosomal enzymes leading to accumulation of undigested macromolecules within the lysosomes and thus cell dysfunction. The clinical manifestation is heterogeneous and neurological involvement represents a major problem. The correction of the defective gene/protein represents the primary strategy for the treatment of these genetic conditions. However, the clinical translation of these approaches is very challenging because of the difficulty in achieving and maintaining therapeutic threshold levels of the corrective enzyme in targeted tissues (particularly in the brain) avoiding the toxicity associated to the high dosage of either viral vectors or infused corrective enzymes. To address this challenge, I have employed a strategy by which lysosomal enzymes are modified to improve their therapeutic potential. Specifically, I have developed and validated a tool through which is possible to generate gain-of-function variants of lysosomal enzymes that exhibit enhanced catalytic activity and/or increased stability in physiological conditions. I believe that enzyme variants generated by my work may produce a beneficial effect in targeted tissues (particularly in the brain) more efficiently and therefore, at lower doses compared to the respective WT enzymes. Therefore, my data may pave the way for the development of enhanced replacement therapies with improved clinical translationability to treat CNS in multiple LSDs.

Proteases are involved in a high number of diseases, and thus, are relevant targets. For that reason our main goal was the discovery of protease inhibitors as therapeutic agents. We focused our study in four proteases: dipeptidyl peptidase IV (diabetes mellitus type 2), prolyl oligopeptidase (cognitive disorders) and cathepsins L and B (cancer).For the discovery of inhibitors, three strategies were selected: medicinal plant screening, high throughput screening and the characterization of a combinatorial chemistry library. Once accomplished the DPP IV recombinant expression optimization, the protein was studied by means of nuclear magnetic resonance (NMR) in order to obtain information of its dynamism. Since DPP IV is a large protein, a strategy combining selective labeling and the use of TROSY-HSQC experiments was used. From the 14 methionine residues of the protease, 11 of them were detected in the NMR spectra. Then, a study of the inhibitor effect on the NMR spectra of DPP IV was done. Interestingly, the corresponding spectra of DPP IV / inactivator afforded an extra signal. We believe that it is a consequence of a small structural change that the protease suffers after inhibition. Afterwards, we planned to find DPP IV from botanical sources. First, we selected plants that were already reported to have antidiabetic action. Common antidiabetic plants were chosen, as well as Brazilian plants and others from the Traditional Chinese Medicine. Besides, a library of Mediterranean plants was also selected. After, extraction and testing of DPP IV inhibitory activity was done. From our tailored collection, the plant AP-3 was selected for further analysis. After fractionation and purification, two molecules were found to be DPP IV inhibitors. Kinetic experiments of the best one, AP-3-a, demonstrated that it was inhibiting DPP IV in a parabolic manner. Then, AP-3-a inhibition of DPP IV was analysed by NMR. The extra signal that was observed with competitive inhibitors was not present. We hypothesized that the lack of appearance of this signal is a result of the parabolic inhibition of AP-3-a. Then, we planned the identification of POP inhibitors by HTS. Our strategy was based in the use of libraries containing non-toxic compounds and lead-like properties. The assay we selected was FP, which allowed the identification of protein binders by competition with a fluorophore-labelled probe. First, the peptide probes were validated as useful probes for the FP assay. Then, the HTS was carried out. Over the 4,500 tested compounds, 73 hits were found to be POP binders. Later, 37 hits were selected by means of clustering, docking data and FP results in order to be validated as POP inhibitors. The subset of molecules was evaluated by enzymatic assays. Six compounds presenting the highest POP inhibition ration were selected for further study. Finally, two POP inhibitors have been described. HTS-43 is a competitive POP inhibitor and HTS-75 displays a parabolic behaviour. It was the first time that parabolic inhibition is reported for POP. We believe that the existence of a non-competitive site would help in the understanding of the relationship between POP and mental diseases. Finally, a novel peptidyl aryl vinyl sulfone library was tested for its inhibitory activity against cathepsins L and B. Among all the 20 molecules of the library, a potent covalent irreversible cathepsin L inhibitor has been found, PAVS-20. The progress-curve of the pre-incubation time representation allowed the calculation of its inhibition constants. Furthermore, evaluation of subsite preferences was done by docking analysis. This allowed understanding the experimental differences in inhibition constants obtained for similar compounds. Since cathepsins L and B are targets for cancer, molecules of the PAVS library are promising candidates for the development of new anticancer drugs.<br>Las proteasas están involucradas en un alto número de enfermedades y por lo tanto, son dianas terapéuticas relevantes. Por este motivo, nuestra principal meta era el descubrimiento de inhibidores de proteasas cómo agentes terapéuticos. Para ello nuestro estudio en cuatro proteasas: la dipeptidil peptidasa IV, la prolil oligopeptidasa y las catepsinas L y B. Para la búsqueda de inhibidores, se seleccionaron tres estrategias: cribado de plantas medicinales, cribado de alto rendimiento y caracterización de una biblioteca proveniente de la química combinatoria. Una vez se llevó a cabo la expresión recombinante de la DPPIV, la proteína se estudió mediante resonancia magnética nuclear (NMR) para obtener información acerca de su dinamismo. Dado que es una proteína grande, se utilizó una estrategia en la que se combinó el marcaje selectivo y el uso de experimentos TROSY-HSQC. Posteriormente, se realizó el estudio de la DPP IV en presencia de sus inhibidores, para observar como estos afectan a la estructura proteica. Después, se realizó la búsqueda de inhibidores de la DPP IV a partir de extractos de plantas medicinales. De nuestra colección, se seleccionó el extracto de la planta AP-3 para un análisis en profundidad. Se detectaron dos inhibidores de la proteasa. El más potente, AP-3-a, se caracterizó cómo un inhibidor parabólico. Después se llevó a cabo el estudio del complejo DPP IV/AP-3-a por NMR. En cuanto a la POP, se realizó la búsqueda de inhibidores mediante cribado de alto rendimiento (HTS). De los 4,500 compuestos testados se obtuvo un total de 73 hits en el ensayo de polarización de la fluorescencia (FP). La validación de estas moléculas mediante docking, clustering y ensayos enzimáticos, permitió identificar seis potentes inhibidores de POP. Uno de ellos, HTS-75, se caracterizó como un inhibidor parabólico. Esta es la primera vez que se describe un inactivador de este tipo para POP. Finalmente, en cuanto a las catepsinas L y B, se cribó una librería de peptidil aril vinil sulfonas. Entre las 20 moléculas testadas se encontró un potente inhibidor irreversible de la catepsina L, el PAVS-20. Además, se realizó un estudio de docking que permitió evaluar las preferencias de los subsitios de las dos proteasas.

Part I. Recently there has been an alarming rise in the number of infections due to pathogenic organisms that are insensitive to our current arsenal of pharmaceuticals, necessitating the identification of new antimicrobial targets. Here, we describe structural studies of two enzymes from the aspartate family of biosynthetic enzymes in order to assess their potential for drug targeting. Our first report details how an unlikely inhibitor with low millimolar binding characteristics, 5-hydroxy-4-oxo-norvaline, can effectively inactivate homoserine dehydrogenase (HSD) through the formation of a tight-binding covalent adduct. This is followed by the structural characterization of the first phenolic-based non-amino acid inhibitor of HSD, which is shown to occupy the substrate binding site and make specific contacts with residues involved in catalysis. Together, these studies lay the foundation for further structure-based drug design of novel inhibitors of HSD. Following this, we report the first structure of the next enzyme in the aspartate pathway, homoserine transacetylase (HTA). As the committal step in microbial methionine biosynthesis, this enzyme is essential to microbial survival. The high-resolution crystal structure identifies HTA as a new member of a larger structural family of enzymes known as the alpha/beta hydrolases. Using the structure as a guide, we propose a rationale for the previously reported ping-pong reaction mechanism for this enzyme. We conclude with a preliminary look into how the natural substrate, homoserine, binds in the active site and some subtle differences between HTAs from different sources.<br>Part II. Microbes have long been admired for their ability to process virtually any chemical. In Part II of this work we will engage in the structural characterization of two enzymes involved in the microbial degradation of xenobiotic compounds. The first enzyme, cyclohexylamine oxidase (CHAO), is the enabling step in the bacterial metabolism of cyclohexylamine. Preliminary crystallographic analysis of cofactor bound and ternary complexes of CHAO, will be used to highlight differences between this enzyme and its closest human homologue. This information will then be used to direct structure inspired mutagenesis studies in order to better understand the substrate specificity of this enzyme and how it might be altered. Finally, the last enzyme to be discussed in this work will be cyclohexanone monooxygenase (CHMO). Also from the same biodegradation pathway as CHAO this enzyme is responsible for the stereo- and regio- specific conversion of small cyclic ketones into lactones. This reaction, known as the Baeyer-Villiger Oxidation, is of tremendous importance in the pharmaceutical industry as lactones often serve as the building blocks of other larger compounds. Surprisingly, despite many years of research there has never been a published report on the structure of this enzyme. We detail here, for the first time, the crystallographic structure of CHMO in multiple conformations. Together, the various structures of CHAO and CHMO presented will provide insight into how bacteria are able to process xenobiotics and how we might use this information for structure based protein design.<br>Partie I. Récemment, il y a eu une augmentation alarmante du nombre d'infections causées par des organismes pathogènes qui sont insensibles aux produits pharmaceutiques existants, d'où la nécessité d'identifier de nouvelles cibles antimicrobiennes. Ici, nous décrivons des études structurales de deux enzymes de la famille des enzymes de biosynthèse de l'aspartate afin d'évaluer leur potentiel en tant que cible pharmaceutique. Notre première étude décrit comment un improbable inhibiteur avec une liaison de l'ordre du millimolaire, 5-hydroxy-4-oxo-norvaline, peut inactiver l'homosérine déshydrogénase (HSD) par la formation d'un complexe covalent stable. L'étude est suivie par la caractérisation structurale du premier inhibiteur de l'HSD qui est dérivé du phénol et non d'un acide aminé, et dont nous démontrons qu'il occupe le site de liaison du substrat et forme des contacts spécifiques avec les résidus impliqués dans la catalyse. Mis ensembles, ces études jettent les bases pour la conception de nouveaux adjuvants de l'HSD basée sur la structure. Suite à cela, nous rapportons la première structure de l'enzyme suivante dans la voie métabolique de l'aspartate, l'homosérine transacétylase (HTA). Cette enzyme est essentielle à la survie microbienne, car elle catalyse une réaction déterminante de la biosynthèse microbienne de la méthionine. La structure cristalline à haute résolution identifie l'HTA comme un nouveau membre d'une large famille structurale d'enzymes appelées les alpha/beta hydrolases. En utilisant la structure comme guide, nous proposons une explication pour le mécanisme de réaction ping-pong rapporté antérieurement pour cette enzyme. Nous concluons avec une perspective sur la façon dont le substrat naturel, l'homosérine, lie le site actif, ainsi que sur les différences subtiles qui existent entre les HTA provenant de sources différentes.<br>Partie II. Les microbes ont depuis longtemps été admirés pour leur capacité à transformer littéralement n'importe quel composé chimique. Dans la deuxième partie de cette thèse, nous entreprenons la caractérisation structurale de deux enzymes impliquées dans la dégradation microbienne des composés xénobiotiques. La première enzyme, la cyclohexylamine oxidase (CHAO), est l'étape déterminante dans le métabolisme bactérien de la cyclohexylamine. L'analyse cristallographique préliminaire de CHAO lié avec un co-facteur ou en complexe ternaire sera utilisée pour souligner les différences entre cette enzyme et son homologue le plus près chez l'humain. Cette information sera ensuite utilisée pour diriger des études de mutagénèse inspirées de la structure afin de mieux comprendre la spécificité de l'enzyme envers son substrat et comment celle-ci pourra être altéré. Enfin, la dernière enzyme dont nous discuterons dans ce travail sera la cyclohexanone monooxygenase (CHMO). Provenant de la même voie métabolique de dégradation que la CHAO, cette enzyme est responsable pour la conversion stéréo- et régio-spécifique de petites cétones cycliques en lactones. Cette réaction, connu sous le nom d'oxydation Baeyer-Villiger, est d'une importance capitale pour l'industrie pharmaceutique car les lactones servent de précurseurs pour la synthèse d'autres composés plus grands. Étonnamment, en dépit de nombreuses années de recherches, il n'y a jamais eu de publication rapportant la structure de cette enzyme. Ici, nous décrivons pour la première fois la structure cristallographique de la CHMO dans des conformations multiples. Ensemble, les structures de CHAO et CHMO présentées ici nous permettront de mieux comprendre comment les bactéries transforment les composés xénobiotiques et comment cette information pourra être utilisé pour l'ingénierie de protéine basée sur la structure. fr

Les glycosyltransférases (GTs) sont une famille importante d’enzymes responsable de la biosynthèse des chaînes de glycosaminoglycane (GAG) des protéoglycanes, composants clés de la matrice extracellulaire et de la membrane plasmique cellulaire impliqués dans la communication, l'adhésion, la migration et la prolifération cellulaires. Les GTs jouent donc un rôle central dans de nombreux processus physiologiques et physiopathologiques tels que les cancers ou encore les maladies dégénératives et génétiques. Parmi ces GTs, la ß1,4-galactosyltransférase 7 (ß4GalT7) est une cible thérapeutique potentielle puisqu'elle : i) catalyse une étape précoce et majeure de la biosynthèse des chaînes de GAG, ii) est impliquée dans une forme rare de maladie génétique des tissus conjonctifs, le syndrome d’Ehlers-Danlos, iii) prend en charge des xylosides exogènes modulant son activité in vitro et in vivo. Ce travail de thèse s'organise sur une étude structure/fonction de cette enzyme afin de cerner les résidus d'acides aminés clés dans l'interaction de l'enzyme avec un substrat modèle, le 4-methylombelliferyl-ß-D-xylose (4-MOX). Les résidus Y194, Y196 et Y199 ont ainsi été identifiés comme clés dans l'architecture du site de fixation du substrat accepteur et dans l'interaction de l'enzyme avec le xyloside. Au contraire, les résidus H195, R226 et le résidu R270, muté dans la forme progéroïde du syndrome d'Ehlers-Danlos, apparaissent comme des résidus « modulant » l'activité de l'enzyme, notamment du fait d'interactions moléculaires impliquant leur squelette peptidique pour H195 et R226 et une boucle flexible pour R270. Ces travaux ont permis de guider la synthèse d’analogues xylosidiques visant à inhiber l'activité de la ß4GalT7 humaine. Parmi les propositions, un dérivé fluoré du 4-MOX apparaît comme un inhibiteur efficace de la ß4GalT7 in vitro et in cellulo. Faiblement cytotoxique, ce dérivé réduit la prolifération des cellules de lignées cancéreuses SW1353 et MB MDA 231. Ces résultats ouvrent la perspective de nouvelles stratégies thérapeutiques utilisant les xylosides comme agents potentiels dans le traitement de cancers ou encore des maladies génétiques des tissus conjonctifs<br>Glycosyltransferases (GTs) are an important family of enzymes involved in the biosynthesis of glycosaminoglycan (GAG) chains of proteoglycans which are key components of cell plasma membranes and of the extracellular matrix, and are thus implicated in cell communication, adhesion, migration and proliferation. GTs are thus key players in numerous pathophysiological processes such as cancers, degenerative and genetic diseases. Among these GTs, ß1,4-galactosyltransférase 7 (ß4GalT7) is a potential therapeutic target since : i) it catalyzes a rate-limiting step in the early phase of the GAG chains biosynthesis, ii) it is implicated in a rare genetic connective tissue disorder (Ehlers-Danlos Syndrome), iii) its in vitro and in vivo activity can be modulated by exogenous xyloside molecules. This PhD work is focused on a structure/function study of the enzyme aiming to identify key amino acid residues that interact with 4-methylumbelliferyl-ß-D-xylose (4-MUX), taken as reference substrate. Y194, Y196 and Y199 have been identified as key residues for the architecture of the acceptor substrate binding site and establish interactions with 4-MUX. By contrast, H195, R226 and R270, a residue mutated in the progeroid form of Ehlers-Danlos Syndrome, should rather be considered as “modulating” residues towards the ß4GalT7 activity, H195 and R226 interacting with 4-MUX with their polypeptide backbone, and R270 via a flexible loop. This work guided the design of xylosidic compounds that would potentially inhibit the ß4GalT7 activity. Thus, a fluorinated derivative of 4-MUX appeared as an efficient in vitro and in cellulo inhibitor of the enzyme. Poorly cytotoxic, this compound also reduced the proliferation rate of cancer cells SW1353 and MB MDA 231. Altogether, these results offer new therapeutic strategies using xylosides as potential therapeutic agents in the treatment of cancer or rare genetic disorders

Thesis (MSc)--Stellenbosch University, 2015.<br>ENGLISH ABSTRACT: In this study, the influence of rooibos on the catalytic activity of enzymes 17β -hydroxysteroid dehydrogenase type 3 (17βHSD3), 17β-hydroxysteroid dehydrogenase type 5 (AKR1C3), 17β-hydroxysteroid dehydrogenase type 2 (17βHSD2), 5α-reductase type 1 (SRD5A1) and 5α-reductase type 2 (SRD5A2), which catalyse prostate androgen metabolism, was investigated. The activities of both 17βHSD3 and AKR1C3 heterologously expressed in CHO-K1 and HEK293 cells were inhibited significantly by rooibos, with rooibos reducing the conversion of androstenedione (A4) and 11keto-androstenedione (11KA4) to testosterone (T) and 11ketotestosterone (11KT), respectively. The catalytic activity of 17βHSD2 towards T, 11hydroxytestosterone (11OHT) and 11KT was also significantly inhibited by rooibos in transiently transfected HEK293 cells. In transiently transfected HEK293 cells rooibos did not inhibit SRD5A1 while the rate of T conversion to dihydrotestosterone (DHT) by SRD5A2 was decreased. Analysis of steroid metabolism in PNT2 cells also suggests that rooibos does not modulate the catalytic activity of endogenously expressed SRD5A towards A4, however, the conversion of T to DHT was reduced. In addition, reductive 17βHSD activity towards A4 was inhibited in the presence of rooibos in both PNT2 and BPH-1 cells. In contrast, the conversion of 11KA4 to 11KT was inhibited in BPH-1, PC-3 and LNCaP cells, with negligible conversion of 11KA4 in PNT2 cells. Interestingly, data suggests inhibition of 3α-hydroxysteroid dehydrogenase type 3 (AKR1C2) activity in the production of androsterone (AST) from 5α–androstenedione (5α-dione), as well as the dehydrogenase reaction of T to A4 in PNT2 cells by rooibos. Androgen metabolism pathways were subsequently investigated in LNCaP cells to determine androgen metabolism by endogenous enzymes. Rooibos resulted in the reduced conversion of A4 in LNCaP cells to the same extent as indomethacin, a known AKR1C3 inhibitor. Rooibos also modulated T, DHT and AST metabolism in LNCaP cells. Furthermore, uridine diphosphate glucuronosyltransferase (UGT) activity in LNCaP cells was inhibited by rooibos, decreasing T-, DHT– and AST-glucuronide formation. These data prompted subsequent investigations into the influence of rooibos at cellular level, and prostatespecific antigen (PSA) levels were assayed in the presence of rooibos. PSA was significantly inhibited by rooibos in the absence and presence of DHT, suggesting possible interaction of rooibos with the mutated androgen receptor (AR) or estrogen receptor-β (ERβ) expressed in LNCaP cells. Taken together, rooibos inhibited the catalytic activity of key enzymes that catalyse the activation of androgens in the prostate, as well as inhibiting enzymes involved in the conjugation of androgens. At cellular level, PSA levels were also decreased by rooibos, possibly through AR or ERβ interactions – clearly indicating a modulatory role for rooibos in active androgen production.<br>AFRIKAANSE OPSOMMING: In hierdie studie was die invloed van rooibos ten opsigte van die katalitiese aktiwiteite van die ensieme 17β-hidroksi-steroïed-dehidrogenase tipe 2, tipe 3 en tipe 5 (17βHSD2, 17βHSD3, AKR1C3), asook 5α-reduktase tipe 1 en tipe 2 (SRD5A1, SRD5A2) ondersoek. Hierdie ensieme is betrokke in die produksie van androgene in die prostaat. Rooibos het die katalitiese aktiwiteit van 17βHSD3 en AKR1C3 in CHO-K1 en HEK293 selle beïnvloed en het vermindere omskakeling van androstenedioon (A4) en 11keto-androstenedioon (11KA4) na testosteroon (T) en 11-ketotestosteroon (11KT), afsonderlik, veroorsaak. Die katalitiese aktiwiteit van 17βHSD2 teenoor T, 11-hidroksie-testosteroon (11OHT) en 11KT was ook beïnvloed in die teenwoordigheid van rooibos in HEK293 selle. Die katalitiese aktiwiteit van SRD5A1 teenoor A4 en T is nie beïnvloed deur rooibos nie, alhoewel dit voorkom asof rooibos die omsettingstempo van T na dihidrotestosteroon (DHT) deur SRD5A2, getransfekteer in HEK293 selle, verminder het. Verdere ondersoeke is in normale prostaat epiteel selle, in die teenwoordigheid van rooibos uitgevoer. Rooibos het geen invloed op die katalitiese aktiwiteit van SRD5A teenoor A4 gehad nie, alhoewel vermindere omskakeling van T na DHT aangetoon kon word. Rooibos het ook die omskakeling van A4 na T in beide PNT2 en BPH-1 selle tot „n mate geïnhibeer. Die omskakeling van 11KA4 na 11KT was ook verminder in BPH-1, PC-3 en LNCaP selle. Die omskakeling van 11KA4 na 11KT was beduidend laer in PNT2 selle en kon die invloed van rooibos nie aangetoon word nie. Bykomende data toon dat rooibos ook die omskakeling van 5α-androstenedioon (5α-dione) na androsteroon (AST), gekataliseer deur 3α-hidroksi-dehidrogenase tipe 3 (AKR1C2), verminder, gesamentlik met die vermindere omskakeling van T na A4, deur 17βHSD2, in PNT2 selle. Hierdie studie het ook ondersoek ingestel, na die metabolisme van androgene in LNCaP selle. Vermindere A4 metabolisme is in die teenwoordigheid van rooibos asook in die teenwoordigheid van indometasien, „n bekende AKR1C3 inhibitor, gevind. Rooibos verminder dus die aktiwiteit van reduktiewe 17βHSD in LNCaP selle. Verandering in die metabolisme van T, DHT en AST in LNCaP selle, in die teenwoordigheid van rooibos, is ook gevind. Verdere ondersoek in LNCaP selle het gewys dat rooibos „n vermindering in die produksie van gekonjugeerde T, DHT en AST veroorsaak. Die studie het die invloed van rooibos op prostaat-spesifieke antigeen (PSA) ook ondersoek. Daar is vasgestel dat rooibos die vlakke van PSA verminder in die afwesigheid en teenwoordigheid van DHT in LNCaP selle. Hierdie resultaat dui op moontlike interaksie van rooibos met die androgeen (AR) of estrogeen-reseptor-β (ERβ), teenwoordig in LNCaP selle. Rooibos het die katalitiese aktiwiteit van ensieme, wat bydra tot androgeen produksie, geïnhibeer, asook die konjugasie van androgene. Op „n sellulêre vlak, het rooibos die vlakke van PSA-sekresie verminder, wat moontlike interaksie met die AR en ERβ aandui. Hierdie bevindings dui daarop dat rooibos wel n rol het om te speel in die modulasie van aktiewe androgene in die prostaat.

Thesis (PhD)--Stellenbosch University, 2015.<br>ENGLISH ABSTRACT: This study describes: • the influence of a methanolic extract of unfermented Rooibos and five major Rooibos flavonoids, aspalathin, nothofagin, rutin, orientin and vitexin, on the activities of key adrenal steroidogenic enzymes - cytochrome P450 17β- hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase • the development of a novel UPLC-MS/MS method for the separation and quantification of 21 adrenal steroid metabolites; • the influence of Rooibos and aforementioned flavonoids on adrenal steroid hormone production in H295R cells - a human adrenal carcinoma cell line expressing the enzymes catalysing the production of mineralocorticoids, glucocorticoids and adrenal androgens, assayed under both basal (normal) and forskolin-stimulated (stressed) conditions; • the influence of Rooibos on the inter-conversion between cortisol and cortisone by 11βHSD1 and 11βHSD2 expressed in CHO-K1 cells; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male Wistar rats; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male and female human test subjects at risk for developing cardiovascular disease. (3βHSD2), cytochrome P450 21-hydroxylase (CYP21A2) and cytochrome P450 11β-hydroxylase (CYP11B1), expressed in non-steroidogenic COS-1 cells;<br>AFRIKAANSE OPSOMMING: Hierdie studie beskryf: • die invloed van metanoliese ekstrakte van ongefermenteerde Rooibos en vyf van die hoof flavonoïedverbindings in Rooibos, aspalatien, notofagien, rutien, oriëntien en viteksien, op die aktiwiteite van ensieme wat steroïedbiosintese in die bynier kataliseer – sitochroom P450 17α-hidroksilase/17,20-liase (CYP17A1), 3β-hidroksisteroïed dehidrogenase (3βHSD2), sitochroom P450 21-hidroksilase (CYP21A2) en sitochroom P450 11β-hidroksilase (CYP11B1), uitgedruk in nie-steroïed produserende COS-1 selle; • die ontwikkeling van ‘n geskikte UPLC-MS/MS metode vir die skeiding en kwantifisering van 21 steroïedmetaboliete in die bynier; • die invloed van Rooibos en die bg. flavonoïede op steroïedproduksie in H295R selle – ‘n menslike bynier kanker sellyn gekenmerk deur die ekspressie van die steroidogeniese ensieme wat die produksie van mineralokortikoïede, glukokortikoïede en bynierandrogene kataliseer, geanaliseer onder beide basale (normale) en forskoliengestimuleerde (gestresde) kondisies; • die invloed van Rooibos op die omeenskakeling tussen kortisol en kortisoon deur 11βHSD1 and 11βHSD2 in CHO-K1 selle; • die invloed van Rooibosinname op vlakke van sirkulerende steroïed hormone en relatiewe verhoudings in die bloed van manlike Wistarrotte; • die invloed van Rooibosinname op sirkulerende steroïed hormoon vlakke en relatiewe verhoudings in die bloed van mans en vrouens met ‘n hoë risiko vir die ontwikkeling van kardiovaskulêre siektes.

La synthèse guidée par la cible de ligands protéiques est une stratégie innovante pour découvrir des composés bioactifs. En particulier, la Kinetic Target-Guided Synthesis (KTGS) and the Dynamic Combinatorial Chemistry (DCC) ont permis, ces dernières années, de découvrir des ligands originaux pour des cibles thérapeutiques mal explorées, ce qui a permis de lancer des projets de découverte de médicaments. Ce projet de thèse vise à utiliser la KTGS pour découvrir, puis optimiser des ligands de deux classes de métalloenzymes que sont les aminopeptidases du réticulum endoplasmiques (ERAP) et l’élastase LasB de la bactérie Pseudomonas aeruginosa. Les ERAPs (1 et 2) participent au processus de maturation des antigènes. Ces enzymes clivent les précurseurs peptidiques en peptides antigéniques matures afin que ceux-ci disposent d’une taille optimale pour leur complexation au complexe majeur d’histocompatibilité de classe I et ainsi initient ou non la réponse immunitaire adaptative. Les niveaux d’expression de ces protéases ainsi que des polymorphismes d’un seul nucléotide ont été associé au développement de cancers et de maladies auto-immunes. Ainsi, la modulation de ces enzymes permettrait de lutter contre les pathologies associées au système immunitaire. P. aeruginosa est une bactérie Gram negative dotée d’une virulence et d’une résistance aux antimicrobiens remarquable. Aujourd’hui, la résistance aux antibiotiques représente un enjeu de santé publique majeur et il y a un besoin urgent en nouvelles thérapeutiques. Afin de satisfaire ce besoin, de nouvelles stratégies sont apparues comme celle consistant à cibler la virulence des bactéries afin de « désarmer » celles-ci. LasB représente une cible thérapeutique de choix de par sa localisation extracellulaire et ses implications physiopathologiques (colonisation, invasion, évasion à la réponse immunitaire, formation de biofilm, etc.). Bien qu'il y ait un besoin médical évident non satisfait dans ces deux aires thérapeutiques, aucun modulateur des ERAPs ni de LasB n'a atteint le marché. Ainsi, l’utilisation de la stratégie KTGS suivie de phases d’optimisation nous ont permis d’identifier et optimiser de nouvelles familles de ligands de ces enzymes. Ces composés peuvent être considérés comme des leads prometteurs puisqu’ils présentent des affinités nanomolaires pour leurs cibles respectives, des profils de sélectivité et de toxicité ainsi que des propriétés physicochimiques remarquables<br>Target-guided synthesis of protein ligands is an innovative strategy to discover bioactive compounds. In particular, the Kinetic Target-Guided Synthesis (KTGS) and the Dynamic Combinatorial Chemistry (DCC) have allowed, in recent years, the discovery of novel ligands for poorly explored therapeutic targets, which has enabled drug-discovery projects. This thesis project aims at using KTGS to discover and optimize ligands for two classes of metalloenzymes, namely endoplasmic reticulum aminopeptidases (ERAPs) and elastase LasB from the bacterium Pseudomonas aeruginosa. ERAPs (1 and 2) are involved in the process of antigen maturation. These enzymes cleave peptide precursors into mature antigenic peptides so that they have an optimal size for their complexation to the major histocompatibility complex of class I and thus initiate or not the adaptive immune response. The expression levels of these proteases as well as single nucleotide polymorphisms have been associated with the development of cancers and autoimmune diseases. Thus, the modulation of these enzymes would allow to fight against pathologies associated with the immune system. P. aeruginosa is a Gram-negative bacterium with remarkable virulence and antimicrobial resistance. Today, antibiotic resistance represents a major public health issue and there is an urgent need for new therapeutics. In order to meet this need, new strategies have emerged such as targeting the virulence of bacteria to "disarm" them. LasB represents a therapeutic target of choice due to its extracellular localization and its physiopathological implications (colonization, invasion, evasion of immune response, biofilm formation, etc.). Although there is a clear unmet medical need in these two therapeutic areas, no modulator of ERAPs or LasB has reached the market. Thus, the use of the KTGS strategy followed by optimization phases allowed us to identify and optimize new families of ligands for these enzymes. These compounds can be considered as promising lead compounds since they present nanomolar affinities for their respective targets, selectivity and toxicity profiles as well as remarkable physicochemical properties

Adenosine deaminase acting on transfer RNA (ADAT) is a human heterodimeric enzyme that catalyzes the deamination of adenosine (A) to inosine (I) at the first position of the anticodon of transfer RNAs (tRNAs) (position 34, or wobble position); one of the few essential post-transcriptional modifications on tRNAs (1-5). Inosine 34 allows the recognition of three different nucleotides: cytidine, uridine and adenosine, at the third position of the codon, thus increasing the decoding capacity of tRNAs to more than one messenger RNA (mRNA) codon (adenosine 34 can in principle only pair with codons with uridine at the third position) (6, 7). This alters the tRNA pool available for each codon and it has been proved to align the correlation between codon usage and tRNA gene copy number (8). It has also been suggested to improve fidelity and efficiency of translation (8, 9), especially for mRNAs enriched in codons translated by modified tRNAs (10, 11). Monitoring ADAT-mediated deamination is crucial for the characterization of the enzyme in terms of activity, substrates, regulation, as well as for drug discovery purposes. However, this analysis is often challenging, laborious and lacks quantitativeness. We developed an in vitro deamination assay based on restriction fragment length polymorphism (RFLP) analyses to monitor ADAT activity in an efficient, cost-effective, and semiquantitative manner (12). To overcome a limitation of the method being the need of reverse transcription and amplification of the tRNA, we designed a direct method to quantify I34 formation in vitro using the first fluorescent analogs of nucleic acids that have been reported to undergo enzymatic deaminations (13-15). ADAT has been conserved over the evolution with the acquisition of multi-substrate specificity. Whereas its bacterial homolog TadA deaminates exclusively tRNAArg (2), the human enzyme deaminates eight different tRNAs (3, 16). However, the mechanisms that drove this evolution remain unknown. While the substrate recognition in TadA has been well studied, in the eukaryotic ADAT is poorly understood. Through in vitro enzymatic activity assays with different variants of tRNAArg and tRNAAla, we elucidated the most important features for efficient A34-to-I34 conversion and characterized the substrate recognition of the human enzyme. We also proposed a new potential mechanism of control of ADAT deamination activity by human tRNA-derived fragments, which provides new insights into the regulation of ADAT function and may open a door for the development of new strategies to modulate ADAT activity. A missense mutation (V128M) in one of the two subunits of the human ADAT enzyme causes intellectual disability and strabismus, but the molecular bases of the pathology are unknown (17, 18). We characterized human ADAT in terms of kinetics and structure, and investigated the effect of the V128M mutation. We found that this substitution decreases ADAT deamination activity, and severely affects the stability of the quaternary structure of the enzyme. In this regard, we discovered small molecules with the ability to activate the enzyme, which could potentially recover the defective tRNA editing caused by the mutation. References 1. Gerber AP, Keller W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 1999;286(5442):1146-9. Epub 1999/11/05. 2. Wolf J, Gerber AP, Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. The EMBO journal. 2002;21(14):3841-51. Epub 2002/07/12. 3. Torres AG, Pineyro D, Rodriguez-Escriba M, Camacho N, Reina O, Saint-Leger A, et al. Inosine modifications in human tRNAs are incorporated at the precursor tRNA level. Nucleic acids research. 2015;43(10):5145-57. Epub 2015/04/29. 4. Zhou W, Karcher D, Bock R. Identification of enzymes for adenosine-to-inosine editing and discovery of cytidine-to-uridine editing in nucleus-encoded transfer RNAs of Arabidopsis. Plant physiology. 2014;166(4):1985-97. Epub 2014/10/16. 5. Tsutsumi S, Sugiura R, Ma Y, Tokuoka H, Ohta K, Ohte R, et al. Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast. J Biol Chem. 2007;282(46):33459-65. Epub 2007/09/19. 6. Crick FH. Codon--anticodon pairing: the wobble hypothesis. Journal of molecular biology. 1966;19(2):548-55. Epub 1966/08/01. 7. Torres AG, Pineyro D, Filonava L, Stracker TH, Batlle E, Ribas de Pouplana L. A-to-I editing on tRNAs: biochemical, biological and evolutionary implications. FEBS Lett. 2014;588(23):4279-86. Epub 2014/09/30. 8. Novoa EM, Pavon-Eternod M, Pan T, Ribas de Pouplana L. A role for tRNA modifications in genome structure and codon usage. Cell. 2012;149(1):202-13. Epub 2012/04/03. 9. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002;84(8):791-803. Epub 2002/11/30. 10. Rafels-Ybern A, Attolini CS, Ribas de Pouplana L. Distribution of ADAT-Dependent Codons in the Human Transcriptome. International journal of molecular sciences. 2015;16(8):17303- 14. Epub 2015/08/01. 11. Rafels-Ybern A, Torres AG, Grau-Bove X, Ruiz-Trillo I, de Pouplana LR. Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla. RNA biology. 2017:0. Epub 2017/09/08. 12. Wulff TF, Arguello RJ, Molina Jordan M, Roura Frigole H, Hauquier G, Filonava L, et al. Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method. Biochemistry. 2017;56(31):4029-38. Epub 2017/07/14. 13. Sinkeldam RW, McCoy LS, Shin D, Tor Y. Enzymatic interconversion of isomorphic fluorescent nucleosides: adenosine deaminase transforms an adenosine analogue into an inosine analogue. Angew Chem Int Ed Engl. 2013;52(52):14026-30. Epub 2013/11/30. 14. McCoy LS, Shin D, Tor Y. Isomorphic emissive GTP surrogate facilitates initiation and elongation of in vitro transcription reactions. Journal of the American Chemical Society. 2014;136(43):15176-84. Epub 2014/09/26. 15. Rovira AR, Fin A, Tor Y. Chemical Mutagenesis of an Emissive RNA Alphabet. J Am Chem Soc. 2015;137(46):14602-5. Epub 2015/11/03. 16. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic acids research. 2009;37(Database issue):D159-62. Epub 2008/10/30. 17. Alazami AM, Hijazi H, Al-Dosari MS, Shaheen R, Hashem A, Aldahmesh MA, et al. Mutation in ADAT3, encoding adenosine deaminase acting on transfer RNA, causes intellectual disability and strabismus. Journal of medical genetics. 2013;50(7):425-30. Epub 2013/04/27. 18. El-Hattab AW, Saleh MA, Hashem A, Al-Owain M, Asmari AA, Rabei H, et al. ADAT3- related intellectual disability: Further delineation of the phenotype. American journal of medical genetics Part A. 2016;170A(5):1142-7. Epub 2016/02/05.<br>L’adenosina deaminasa específica per RNA de transferència (ADAT) és un enzim humà heterodimèric que catalitza la reacció de deaminació de l’adenosina (A) a inosina (I) a la primera posició de l’anticodó de RNAs de transferència (tRNAs) (també anomenada posició 34 o posició de balanceig); una de les poques modificacions post-transcripcionals essencials en tRNAs (1-5). La inosina 34 permet el reconeixement de tres nucleòtids diferents: citidina, uridina i adenosina, a la tercera posició del codó, augmentant per tant la capacitat de descodificació dels tRNAs a més d’un codó en l’RNA missatger (mRNA) (l’adenosina 34 en principi únicament pot aparellar-se amb uridina en la tercera posició) (6, 7). Això altera els nivells de tRNAs disponibles per cada codó i s’ha demostrat que alinea la correlació entre l’ús de codons i número de còpies gèniques de cada tRNA (8). També s’ha suggerit que millora la fidelitat i eficiència de la traducció (8, 9), especialment per mRNAs enriquits en codons traduïts per tRNA modificats (10, 11). Monitoritzar la deaminació produïda per ADAT és clau per la caracterització de l’enzim en termes d’activitat, substrats, regulació, així com també pel disseny de fàrmacs. No obstant, aquest anàlisi és sovint complex, laboriós i poc quantitatiu. Per això, hem desenvolupat un assaig de deaminació in vitro basat en el polimorfisme de longitud dels fragments de restricció (RFLP) amb el propòsit de monitoritzar l’activitat d’ADAT de manera eficient, cost-efectiva i semiquantitativa (12). Per superar una limitació del mètode essent la necessitat de transcripció reversa i amplificació del tRNA, hem dissenyat un mètode directe per quantificar la formació d’I34 in vitro utilitzant els primers anàlegs fluorescents d’àcids nucleics que són substrats de deaminacions enzimàtiques descrits fins al moment (13-15). ADAT ha estat conservat en l’evolució amb l’adquisició de multi-especificitat de substrat. Mentre que el seu homòleg bacterià TadA deamina exclusivament tRNAArg (2), l’enzim humà deamina vuit tRNAs diferents (3, 16). Tot i així, els mecanismes que van conduir a aquesta evolució romanen desconeguts. Mentre que el reconeixement de substrat en TadA es coneix bé, en ADAT eucariòtic ha estat poc estudiat. A través d’assajos enzimàtics in vitro amb diferents variants de tRNAArg i tRNAAla, hem elucidat els trets més importants per a una conversió eficient d’A34 a I34 i hem caracteritzat el reconeixement de substrat per part de l’enzim humà. També proposem un nou potencial mecanisme de control de l’activitat d’ADAT per part de fragments derivats de tRNAs humans, el qual ofereix noves perspectives en la regulació de la funció d’ADAT i que pot obrir la porta al desenvolupament de noves estratègies per modular-ne l’activitat. Una mutació sense sentit (V128M) en una de les dues subunitats en l’enzim ADAT humà s’ha associat a retard mental i estrabisme, encara que les bases moleculars de la patologia es desconeixen (17, 18). Hem caracteritzat ADAT humà en termes de cinètica enzimàtica i estructura, i n’hem investigat l’efecte de la mutació V128M. Hem descobert que la substitució de valina 128 per metionina redueix l’activitat deaminatòria d’ADAT i que altera severament l’estabilitat de l’estructura quaternària de l’enzim. En aquest aspecte, hem descobert molècules amb l’habilitat d’activar l’enzim, la qual cosa podria revertir potencialment la reducció en l’activitat enzimàtica causada per la mutació. References 1. Gerber AP, Keller W. An adenosine deaminase that generates inosine at the wobble position of tRNAs. Science. 1999;286(5442):1146-9. Epub 1999/11/05. 2. Wolf J, Gerber AP, Keller W. tadA, an essential tRNA-specific adenosine deaminase from Escherichia coli. The EMBO journal. 2002;21(14):3841-51. Epub 2002/07/12. 3. Torres AG, Pineyro D, Rodriguez-Escriba M, Camacho N, Reina O, Saint-Leger A, et al. Inosine modifications in human tRNAs are incorporated at the precursor tRNA level. Nucleic acids research. 2015;43(10):5145-57. Epub 2015/04/29. 4. Zhou W, Karcher D, Bock R. Identification of enzymes for adenosine-to-inosine editing and discovery of cytidine-to-uridine editing in nucleus-encoded transfer RNAs of Arabidopsis. Plant physiology. 2014;166(4):1985-97. Epub 2014/10/16. 5. Tsutsumi S, Sugiura R, Ma Y, Tokuoka H, Ohta K, Ohte R, et al. Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast. J Biol Chem. 2007;282(46):33459-65. Epub 2007/09/19. 6. Crick FH. Codon--anticodon pairing: the wobble hypothesis. Journal of molecular biology. 1966;19(2):548-55. Epub 1966/08/01. 7. Torres AG, Pineyro D, Filonava L, Stracker TH, Batlle E, Ribas de Pouplana L. A-to-I editing on tRNAs: biochemical, biological and evolutionary implications. FEBS Lett. 2014;588(23):4279-86. Epub 2014/09/30. 8. Novoa EM, Pavon-Eternod M, Pan T, Ribas de Pouplana L. A role for tRNA modifications in genome structure and codon usage. Cell. 2012;149(1):202-13. Epub 2012/04/03. 9. Schaub M, Keller W. RNA editing by adenosine deaminases generates RNA and protein diversity. Biochimie. 2002;84(8):791-803. Epub 2002/11/30. 10. Rafels-Ybern A, Attolini CS, Ribas de Pouplana L. Distribution of ADAT-Dependent Codons in the Human Transcriptome. International journal of molecular sciences. 2015;16(8):17303- 14. Epub 2015/08/01. 11. Rafels-Ybern A, Torres AG, Grau-Bove X, Ruiz-Trillo I, de Pouplana LR. Codon adaptation to tRNAs with Inosine modification at position 34 is widespread among Eukaryotes and present in two Bacterial phyla. RNA biology. 2017:0. Epub 2017/09/08. 12. Wulff TF, Arguello RJ, Molina Jordan M, Roura Frigole H, Hauquier G, Filonava L, et al. Detection of a Subset of Posttranscriptional Transfer RNA Modifications in Vivo with a Restriction Fragment Length Polymorphism-Based Method. Biochemistry. 2017;56(31):4029-38. Epub 2017/07/14. 13. Sinkeldam RW, McCoy LS, Shin D, Tor Y. Enzymatic interconversion of isomorphic fluorescent nucleosides: adenosine deaminase transforms an adenosine analogue into an inosine analogue. Angew Chem Int Ed Engl. 2013;52(52):14026-30. Epub 2013/11/30. 14. McCoy LS, Shin D, Tor Y. Isomorphic emissive GTP surrogate facilitates initiation and elongation of in vitro transcription reactions. Journal of the American Chemical Society. 2014;136(43):15176-84. Epub 2014/09/26. 15. Rovira AR, Fin A, Tor Y. Chemical Mutagenesis of an Emissive RNA Alphabet. J Am Chem Soc. 2015;137(46):14602-5. Epub 2015/11/03. 16. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic acids research. 2009;37(Database issue):D159-62. Epub 2008/10/30. 17. Alazami AM, Hijazi H, Al-Dosari MS, Shaheen R, Hashem A, Aldahmesh MA, et al. Mutation in ADAT3, encoding adenosine deaminase acting on transfer RNA, causes intellectual disability and strabismus. Journal of medical genetics. 2013;50(7):425-30. Epub 2013/04/27. 18. El-Hattab AW, Saleh MA, Hashem A, Al-Owain M, Asmari AA, Rabei H, et al. ADAT3- related intellectual disability: Further delineation of the phenotype. American journal of medical genetics Part A. 2016;170A(5):1142-7. Epub 2016/02/05.

Cancer involves the dysregulated proliferation and growth of cells throughout the body. C-terminal binding proteins (CtBP) 1 and 2 are transcriptional co-regulators upregulated in several cancers, including breast, colorectal, and ovarian tumors. CtBPs drive oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes. CtBPs encode an intrinsic dehydrogenase activity, utilizing intracellular NADH concentrations and the substrate 4-methylthio-2-oxobutyric acid (MTOB), to regulate the recruitment of transcriptional regulatory complexes. High levels of MTOB inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. While encouraging, a good therapeutic would utilize >100-fold lower concentrations. Therefore, we endeavored to design better CtBP-specific therapeutics. The best of these drugs, 3-Cl and 4-Cl HIPP, exhibit nanomolar enzymatic inhibition and micromolar cytotoxicity and showed that CtBP enzymatic function is subject to allosteric interactions. Additionally, the function of the substrate-binding domain has yet to be examined in context of CtBP’s oncogenic activity. To this end, we created several point mutations in the CtBP substrate-binding pocket and determined key residues for CtBP’s enzymatic activity. We found that a conserved tryptophan in the catalytic domain is imperative for function and unique to CtBPs among dehydrogenases. Knowledge of this and other residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Early work interrogated the importance of these residues in cell migration. Taken together, this work addresses the utility of the CtBP substrate-binding domain as a target for cancer therapeutics.

The Best PhD Thesis in the Faculties of Architecture, Arts, Business &Economics, Education, Law and Social Sciences (University of HongKong), Li Ka Shing Prize, 2008-2009<br>published_or_final_version<br>Medicine<br>Doctoral<br>Doctor of Philosophy

The human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. The resulting chimeric BCR-ABLoncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of CML patients, but is unable to completely eradicate BCR-ABL–expressing leukemic cells, suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. Two major reasons cause the imatinib resistance. The first one is the BCR-ABL kinase domain mutations which inhibit the interaction of BCR-ABL kinase domain with imatinib; the second one is the residual leukemia stem cells (LSCs) in the patients who are administrated with imatinib. To overcome these two major obstacles in CML treatment, new strategies need further investigation. As detailed in Chapter II, we evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble Hsp90 inhibitor, IPI-504, in our BCR-ABL retroviral transplantation mouse model. We found that BCR-ABL mutants relied more on the HSP90 function than WT BCR-ABL in CML. More interestingly, inhibition of HSP90 in CML leukemia stem cells with IPI-504 significantly decreases the survival and proliferation of CML leukemia stem cells in vitro and in vivo. Consistent with these findings, IPI-504 treatment achieved significant prolonged survival of CML and B-ALL mice. IPI-504 represents a novel therapeutic approach whereby inhibition of Hsp90 in CML patients and Ph+ ALL may significantly advance efforts to develop a cure for these diseases. The rationale underlying the use of IPI-504 for kinase inhibitor–resistant CML has implications for other cancers that display oncogene addiction to kinases that are Hsp90 client proteins. Although we proved that inhibition of Hsp90 could restrain LSCs in vitro and in vivo, it is still unclear how to define specific targets in LSCs and eradicate LSCs. In Chapter III, we took advantage of our CML mouse model and compared the global gene expression signature between normal HSCs and LSCs to identify the downregulation of Pten in CML LSCs. CML develops faster when Pten is deleted in Ptenfl/fl mice. On the other hand, Pten overexpression significantly delays the CML development and impairs leukemia stem cell function. mTOR is a major downstream of Pten-Akt pathway and it is always activated or overepxressed when Pten is mutated or deleted in human cancers. In our study, we found that inhibition of mTOR by rapamycin inhibited proliferation and induced apoptosis of LSCs. Notably, our study also confirmed a recent clinical report that Pten has been downregulated in human CML patient LSCs. In summary, our results proved the tumor suppressor role of Pten in CML mouse model. Although the mechanisms of Pten in leukemia stem cells still need further study, Pten and its downstream, such as Akt and mTOR, should be more attractive in LSCs study.

A papaína é uma enzima utilizada em formulações tópicas como agente proteolítico debridante, no tratamento de lesões abertas de grande extensão e queimaduras. Também empregada como agente promotor da permeação cutânea, peeling químico e como agente depilatório progressivo. A estabilidade de formulações contendo enzimas não é facilmente alcançada. No presente trabalho realizou-se a modificação da papaína com polietilenoglicol, visando maior estabilidade. O Teste de Estabilidade Normal de formulações cosméticas incorporadas de papaína não modificada e modificada apresentou um perfil diferenciado para a atividade da enzima modificada, nas diferentes condições de temperatura (5 &#177; 1 &#176;C; 22 &#177; 2 &#176;C, 40 &#177; 2 &#176;C), sendo que a mais adequada para a papaína não modificada foi de 5 &#177; 1 &#176;C e para a modificada foi de 22 &#177; 2,0 &#176;C. Estes resultados confirmam o aumento da estabilidade da papaína modificada e o seu potencial de aplicação em formulações de uso tópico.<br>Papain is an enzyme used in formulations for local application as proteolitic debridant agent, treatment of wound exposed in large extension and burns. It is also applied as promotor agent of cutaneous permeation, chemical peeling and as progressive depilatory agent. The stability of formulations with enzymes is not easily obtained. In this work modification of papain with polyethylenglycol was made in order to obtain more stability. The Test of Normal Stability of cosmetic formulations incorporated with papain not-modified and modified, showed a differentiated profile for modified enzyme in different conditions of temperature (5 &#177; 1 &#176;C; 22 &#177; 2 &#176;C, 40 &#177; 2 &#176;C), being that, the most adequated for papain not-modified was 5 &#177; 1&#176;C and for modified was 22 &#177; 2,0 &#176;C. These results confirm the increase of stability of papain modified and its potential application in formulations for local application.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-09-12T18:28:33Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) aline_bacelar_goncalves.pdf: 5988024 bytes, checksum: 24d09b3ca0a2e58cc7aee0ca7ca2528f (MD5)<br>Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-09-18T12:57:54Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) aline_bacelar_goncalves.pdf: 5988024 bytes, checksum: 24d09b3ca0a2e58cc7aee0ca7ca2528f (MD5)<br>Made available in DSpace on 2017-09-18T12:57:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) aline_bacelar_goncalves.pdf: 5988024 bytes, checksum: 24d09b3ca0a2e58cc7aee0ca7ca2528f (MD5) Previous issue date: 2017<br>Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)<br>O tratamento das leucemias ? desafiador por v?rios aspectos, entre os quais podem ser destacados os efeitos adversos e a obten??o de op??es terap?uticas de alta qualidade e de custos razo?veis. A utiliza??o da enzima L-asparaginase como agente terap?utico, limita a fonte ex?gena de asparagina, da qual as c?lulas malignas dependem para o metabolismo celular e para a sobreviv?ncia. Essa ? uma op??o que oferece menores riscos ao paciente e ?s c?lulas sadias, que s?o capazes de sintetizar este amino?cido. Neste cen?rio o objetivo deste trabalho foi selecionar, entre fungos filamentosos, linhagens produtoras da enzima L-asparaginase. O estudo tamb?m buscou avaliar o efeito da varia??o da fonte de carbono e da raz?o carbono-nitrog?nio no crescimento e na express?o da atividade enzim?tica, a fim de desenvolver meios de cultivo para o processo produtivo. Realizou-se tamb?m um estudo do crescimento apical das tr?s linhagens selecionadas, duas do g?nero Penicillium sp. e uma do g?nero Fusarium sp., em diversos meios de cultivo. O conhecimento gerado sobre as linhagens produtoras e os demais estudos realizados permitiram a obten??o de um meio de cultivo que possibilitou a produ??o enzim?tica em at? 11,45 U.min-1.mL-1 com a linhagem de Fusarium sp.<br>Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016.<br>The treatment of leukemia is challenging in many ways, including the adverse effects and obtaining treatment options of high quality and reasonable cost. The use of L-asparaginase enzyme as a therapeutic agent limits the exogenous source of asparagine, which the malignant cells depend for cellular metabolism and survival. This option offers lower risk to patients and healthy cells, which are able to synthesize this amino acid. Therefore, the objective of this work was to select among filamentous fungi, producing strains of L-asparaginase enzyme. The study also aimed at evaluating the effect of varying the carbon source and carbon-nitrogen ratio in the growth and expression of the enzymatic activity to develop culture media for the production process. It was also carried out a study of the apical growth of the three strains selected, two of the genus Penicillium sp. and one Fusarium sp., cultivated in various culture media. The knowledge about the growth of the strains studied in different nutritional sources and other studies allowed obtaining a culture medium that enabled the enzyme production of 11.45 U.min-1.mL-1 by Fusarium sp.

Among infectious diseases, two large groups have great clinical relevance: parasitic and bacterial infections. Belonging to the first category is malaria, caused by the parasite Plasmodium falciparum. Transmission of Plasmodium parasites between humans and Anopheles mosquitoes is one of the most important contributors to the global impact of malaria and to the difficulties encountered in eliminating this parasite1 . Gametocytogenesis, the process by which merozoites switch from asexual replication to produce male and female gametocytes, represents a critical step in malaria transmission and Plasmodium genetic diversity. Still too little is known about the biochemical events that regulate gametocytogenesis and there are few existing drugs able of inhibiting the gametocytes development and block malaria transmission2 . To encourage drug discovery and research, the non-profit foundation Medicines for Malaria Venture (MMV) has provided a library of 400 compounds that present antimalarial activity in the micromolar range, but their molecular targets and mode of action are not necessarily known3 . Here we describe the medchem investigation of one of the most promising hit compound included in this library, MMV019918. MMV019918 has been highlighted in several in vitro studies for its promising antigametocyte activity coupled to activity against schizonts. On the basis of its structure, the synthesis of a new series of compounds with transmission-blocking activity has been designed. It has been found very interesting the activity of one derivative NF2350 which resulted active also in the standard membrane feeding assay (SMFA), to measure subsequent mosquito infection, and which has an improved toxicological profile compared to MMV019918. A further investigation of NF2350 will allow us to optimize the transmission-blocking activity and to indentify its putative target. Regarding bacterial infections, a critical issue to be addressed is bacterial resistance to antibiotics and in particular to β-lactams. Metallo-β-lactamases (MBL) are a family of enzymes involved in the widespread mechanisms of resistance to beta-lactam antibiotics, The diffusion of MBL-producing isolates of Pseudomonas aeruginosa, a bacterial pathogen of primary relevance for both nosocomial and chronic infections of the respiratory tracts in cystic fibrosis patients, is notably increasing in some specific settings. No clinically useful MBLs inhibitors are currently available in therapy3 Here we will present the design, synthesis, and biological investigation of a series of functionalized 2-arylfuran compounds with sub-micromolar antiplasmodial activity, against both asexual and sexual stages. Furthermore, appropriate decoration of this molecular scaffold allowed to obtain activity against some isoforms of MBL (including IMP-1and NDM-1).

The prolyl oligopeptidase family enzymes are a group of medically significant proteins distributed in all kingdoms of life that have several unifying structural and functional features. Members of the family hydrolyse short peptides, normally no more than 30 amino acids long. This strict substrate specificity is regulated by β-propeller domains that restrict access to the active site. Conformational changes allow access to the active site by small peptides but block access to larger, structured peptides. Mechanistic details such as these were revealed when the structure of prolyl oligopeptidase was solved by X-ray crystallography in 1998. Since then, numerous crystal structures of family members have been solved, increasing understanding of the structure-function relationships of these important enzymes. Many members of the family are either proven or potential drug targets. As such, structural characterization of these enzymes and their interactions facilitates the design of pharmaceutical compounds. With this in mind, this research was undertaken, using X-ray crystallography to further understand the interactions between particular members of the prolyl oligopeptidase family and inhibitor compounds. Four specific, potent inhibitors were visualized in complex with the active site of prolyl oligopeptidase. All four inhibitors follow a similar template which was shown to be successful when one of them advanced to phase 3 clinical trials as a treatment for Alzheimer‟s disease. The structure of oligopeptidase B from Trypanosoma brucei, which is involved in cell invasion leading to Trypanosomiasis, was solved to 2.5Å, revealing a number of interesting features and enabling various avenues of study. Finally, a plant derived glutamyl endopeptidase known to be regulated by endogenous inhibitors was overexpressed, purified to a high degree and shown to be correctly folded and active. Establishing this procedure allows for the commencement of crystal trials, as well as study by other biophysical methods.

Glycosaminoglycans are heterogeneous polysaccharides that mediate important biological functions. There has been considerable interest in deciphering the precise GAG sequences that are responsible for protein interactions. In fact, several GAG oligosaccharides have been discovered to date as targeting proteins with higher level of specificity. Yet, it has been difficult to develop GAG oligosaccharides as drugs. One of the key reasons for this state of art is that GAG synthesis is extremely challenging and is highly structure-specific. Thus, much of the biology and pharmacology of GAG remains unknown and unexploited to date. An alternative approach is to prepare GAG oligosaccharides using enzymatic depolymerization of polymeric GAGs. GAG lyases, including heparinases and chondritinases represent powerful tools that can theoretically generate multiple oligosaccharides in parallel. However, it is difficult to implement such procedures with high consistency. Moreover, GAG lyases can digest GAGs down to disaccharides. A priori, non-polymeric GAGs, or alternatively GAG oligosaccharides containing 4 to 10 residues, would be expected to function better as therapeutic agents because they would be more homogeneous and less non-specific than their polymeric precursors. Thus, we reasoned that immobilization of these enzymes may engineer altered biopolymer processing, which may afford longer oligosaccharides in higher proportions and greater consistency. Heparinase-I and chondroitinase ABC were immobilized on CNBr-activated Sepharose and compared with the free form of the enzyme. Immobilized GAG lyases retained high efficiency of depolymerization over a wide range of pH, temperature and reusability. Most importantly, the immobilized enzyme was found to produce larger proportions of oligosaccharides longer than di- and tetra-saccharides as compared to lyases in the free form. A two dimensional separation involves size exclusion chromatography followed by reversed phase ion-pairing ultra performance liquid chromatography coupled to electrospray ionization mass spectrometry was employed to separate and characterize oligosaccharide structures. We have identified 40 heparin oligosaccharides, including regular and rare structures ranging from dp4 to dp10 and 39 chondroitin sulfate oligosaccharides in high homogeneity and significant yields. Overall, this technology is likely to offer a simple and cost effective route to preparation of larger amounts of sequences that can be expected to bind and modulate protein function.

Activation of pathways resulting in an overexpression of oncoproteins, reliant on cap-dependent translation, or mutations of key proteins in a pathway can be advantageous to cancer cells but creates heightened protein quality control pressure. Because of this, there has been an interest in targeting enzymes involved in protein synthesis and protein quality control: such as the eukaryotic initiation factor, eIF4A, a DEAD-box RNA helicase involved in translation initiation, and p97, an AAA+ chaperone involved in protein quality control. Despite some successes in discovering both eIF4A and p97 inhibitors, many of these compounds have pharmacological setbacks. The work in this dissertation defines new inhibitors of eIF4A and p97 with unique mechanisms of action. As described in chapter 2, we demonstrated that a marine-derived sesquiterpene, elatol, can modulate the ATPase activity of eIF4A. We provide further evidence that this molecule inhibits cap-dependent translation. Because there is no clear consensus on the mechanism of action for elatol, we hypothesized that the mechanism of toxicity attributed to elatol is likely through inhibition of cap-dependent translation initiation by targeting eIF4A. In chapter 3, we adapted a colorimetric assay to identify natural products that modulate the ATPase activity of p97 from which withaferin A (WFA) was identified. Because proteostasis modulation can connect each of the reported modes of action of WFA, we hypothesized that the primary mode of cytotoxic action of WFA is through inhibition of protein quality control machinery. Through medicinal chemistry efforts, we were able to improve WFA's biochemical and cellular activities as well as shifting the activity toward p97 and away from the proteasome. The work described in chapter 4 reports that dehydrocurvularin (DHC) and its chlorinated analogs are covalent modifiers of p97 and that the selectivity toward p97 can be attributed, in part, to the electronic effects of the chlorines. Taken together, this work highlights the significance of targeting protein translation and quality control, by modulation of eIF4A and p97 activity respectively, as promising anticancer therapeutics.

Natural products have been the source of numerous leads for several drugs. As these natural products are often isolated in small quantities, it is necessary to produce them synthetically to allow testing for biological activity. Furthermore, synthesis allows the preparation of unnatural analogues for SAR studies. Cyclic peptides represent an important family of biologically active natural products. The hepta- and octacyclopeptides sanguinamide A and sanguinamide B were recently isolated in submicromolar amounts by the Molinski group. The lack of material prevented biological evaluation of the natural products. For this reason and to confirm the structural elucidation we have targeted the total synthesis of sanguinamides. In addition to two proline residues, sanguinamides A and B include heterocycles and natural L-amino acid residues. We have completed the total syntheses of sanguinamides A and B; however the synthetic rotamers differed in both cases from the natural rotamers. We have investigated the influence of macrocyclisation on cis/trans conformational preference of the proline residues for the synthesis of sanguinamide A. We attempted several isomerisations and calculated the relative energies of the different sanguinamide conformers. [D-Ile]-Sanguinamide A, Cys(tBu) analogue of sanguinamide A and the synthetic sanguinamide B displayed antibacterial activity while the synthetic trans, trans-sanguinamide A displayed mild tyrosine kinase inhibitory activity. While extracted stylissamide A showed inhibition of translation during the elongation step, even though being structurally identical to the natural product, the synthetic compound prepared by macrocyclisation from a linear precursor was found to be totally inactive. Histones undergo different types of covalent modifications on the N-terminal tails such as acetylation, phosphorylation and methylation. Histone modification is a major mechanism of regulation in gene expression, replication and repair. Deregulation of histone modifications leads to cancer progression and therefore, inhibitors of enzymes which are able to catalyse the addition and removal of these epigenetic marks have therapeutic potential for treating cancer. An enzyme of particular interest is the family of zinc-dependent histone deacetylases (HDACs) that remove acetyl groups from acetylated lysine residues. Depsipeptides were prepared as HDAC inhibitors. We will ii present our total synthesis of largazole along with a range of analogues and discuss the SAR obtained from HDAC and cell proliferation assays. We elucidated the stereochemistry of burkholdac B by total synthesis of three diastereomers. The diastereomers made along with the natural product were tested as HDAC inhibitors. We are interested in inhibitors of lysine-specific demethylase 1 (LSD1) which is a different kind of epigenetic enzyme involved in demethylation of histone proteins in chromatin. Tranylcypromine is known to be an LSD1 inhibitor. Analogues of PCPA have been synthesised in order to explore the structure-activity relationships of this inhibitor. Analogues were also prepared and tested as LSD1 inhibitors.

Clustered regularly interspaced short palindromic repeats (CRISPR) are derived from a bacterial and archaeal adaptive immune system. The core enzymes of CRISPR are RNA-guided endonucleases that sequence-specifically cleave foreign double-stranded DNA. Improving and controling the properties of the CRISPR system is a crucial step in advancing the therapeutic potential of CRISPR technology. Several classes of these enzymes exist and are being adapted for biotechnology, such as genome engineering. Cas12a (Cpf1) is a Type V CRISPR-associated (Cas) enzyme that naturally uses only one guide RNA, in contrast to Type II CRISPR-Cas9 enzymes. Thus, Cpf1 may represent a simpler, more practical tool for applications such as gene editing and therapeutics. This dissertation comprises four related studies in this area. To better understand the functional requirements for Cpf1-crRNA interaction and develop modified crRNAs suitable for synthetic biology and therapeutic applications, the first study performed nucleotide substitutions in the crRNA. It focused on the protein-interaction motif of the crRNA by incorporating base changes at the 2ʹ position that alter hydrogen-bonding capacity, sugar pucker, and flexibility. DNA substitutions in RNA can probe the importance of A-form structure, 2ʹ-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. In addition, Chemical modifications include 2'-deoxy, 2'-fluoro, 2'- deoxy-arabinonucleic acid, and oxepane. Our study discovered that 2'-fluoro maintains the A-form structure and is compatible with AsCpf1 activity. Biochemical endonuclease activity, gene editing efficiency, Cpf1 binding affinity, and ribonucleoprotein stability were used to assess the tolerance and effects of modification. Characterizing structure-function requirements for Cpf1-crRNA interaction will facilitate better design and tuning of Cpf1 enzymes. The second study established a FRET-based assay in collaboration with a computational collaborator to identify small molecule inhibitors predicted by virtual docking and simulations. This study aims to lay the foundation for efficient, safe implementation of CRISPR-Cpf1. The third study used chemically modified Cas9-guide RNAs to offset known weaknesses of CRISPRi. It takes advantage of the high binding affinity and nuclease resistance of modified guides to potentially reduce the required components for CRISPRi.

Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterised by GAA deficiency in the cell lysosomes. The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this project was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO clonal cell line and then developed a two-step bioprocess based on capture chromatographic step anion exchange (IEX) and intermediate hydrophobic interaction (HIC). The purity of GAA after HIC was determined via LC/MSMS to be above 80%. We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a Null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison, only 8% of lysosomes in the Null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines [GAA, mAb and Null] capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co-eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase.

Clostridium difficile is the most common cause of hospital acquired infections. While the current treatments of choice, antibiotics, are generally effective in promoting recovery ,the increased incidence of C. difficile infections and treatment failure associated with antibiotic resistance combined with the emergence of hypervirulent strains highlights the need to develop therapeutic approaches that specifically target the pathogen without causing collateral damage to the protective microbiota. Several non-antibiotic approaches are currently being investigated, such as bacteriophage therapy. For this reason, we attempted to isolate C. difficile specific lytic bacteriophages which could form the basis of a treatment for C. difficile. While we were unable to isolate lytic phages, we were able to isolate twelve temperate bacteriophages from twenty-three clinical isolates of C. difficile using mitomycin C. Unfortunately we failed to identify a susceptible host strain capable of supporting the replication of these phages. This failure may in part be due to repeated episodes of phage infection over time, which have resulted in the emergence of “phage resistant” species mediated by systems such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Employing a PCR-based approach using primers specific for the lysin genes of five previously isolated C. difficile phages, we found evidence to suggest that repeated bacteriophage infection is a common event for clinical isolates of C. difficile. Our inability to isolate a lytic bacteriophage prompted us to adopt an alternative approach in which we used endolysin enzyme of five previously identified C. difficile phages as recombinant protein. These lysins showed broad spectrum activity against the vegetative forms of a large collection of C. difficile ribotypes with little or no activity against other species, supporting their potential as therapeutic agents. We also identified a genome associated lysin (CD630, YP_001088405), which lysed vegetative C. difficile in a similar manner to the phage derived lysins. We also cloned and expressed a spore cortex lytic enzyme (SleC) which targeted the cortex of C. difficile spores. Unfortunately this enzyme was inactive against intact spores, suggesting that the outer layers of the spore act as a permeability barrier. The results of this study showed in vitro the applicability of endolysins against the vegetative form of C. difficile and the activity of spore cortex lytic enzyme against coatless spores, offering interesting perspectives for evaluation of the antibacterial activity of a mixture of endolysin and spore cortex lytic enzyme.

Despite intensive research, cancer remains one of the major causes of worldwide morbidity. It is widely believed, however, that if currently available anti-cancer drugs could be delivered specifically to tumours then the disease would have been mastered. The delivery of prodrug converting enzymes by clostridial spores specifically to the anoxic centres of tumours is one potential delivery mechanism. This is due to the extreme selectivity of spores to germinate solely in the hypoxic regions of tumours. Once germinated, the expression of a prodrug converting enzyme converts a systemical1y administered prodrug to a highly toxic drug only in the tumour. Previous studies using Clostridium acetobutylicum and Clostridium beijerinckii as the delivery vehicle highlighted that prodrug converting enzyme expression is only found in tumours. However, no significant anti-tumour affect was observed. Two possible reasons were evolved. Firstly, expression of the prodrug converting enzyme may be low, and/or, secondly, the tumours may not be colonised sufficiently to promote an antitumour effect. Preliminary studies identified that Clostridium sporogenes NCIMB 10696 may represent a more suitable host. Higher spore titres could be prepared and, once administered, higher cell counts are found in the colonised tumours. Prodrug converting enzymes with improved kinetics over pre-existing enzymes have also been identified. Once effective gene transfer systems and expression systems had been developed, suitably high levels of several different prodrug converting enzymes, in particular nitroreductases, were obtained. Initial in vivo studies on one of the early recombinant strains identified a definite anti-tumour effect. Since those initial studies, further improvements to expression have been made. It is hoped that a more significant anti-tumour affect would result from using these improved strains. It is the ultimate aim of CDEPT to have the prodrug converting enzymes integrated into the host genome so as to negate the use of antibiotics. Towards this, studies on the use of both classical and novel integrative technologies have been investigated.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2006.<br>"June 2006."<br>Includes bibliographical references.<br>Glycans are chemically heterogeneous macromolecules that have profound importance in a variety of biological processes. Located at the surfaces of cells, deposited in the extracellular matrix, or attached to soluble signaling agents, these molecules are characterized by a structural complexity that has thus far prevented their widespread exploitation in biomedicine. Insight into the fine structure and sequence of these complex biomolecules provides a novel niche for the development of therapeutic interventions. Such an understanding is rapidly accumulating via the molecular cloning, recombinant genetic expression, and protein purification of glycosaminoglycan-degrading enzymes. To this end, enzymes from the chondroitinase family of Pedobacter heparinus and Proteus vulgaris were developed. This library of enzymatic tools will reveal glycoconjugate structure-function motifs, allowed for by structural elucidation of glycan species using coupled bioinformatics/analytical chemistry techniques centered on mass spectrometry, nuclear magnetic resonance, and capillary electrophoresis.<br>(cont.) Biochemical characterization of these enzymes has allowed for the rational genetic manipulation of substrate recognition and binding site amino acid residues, producing site-directed protein engineered mutants with altered action pattern and substrate specificity. Progress in these areas will allow for the elucidation of critical roles of glycans in the biological regulation of growth factors, morphogens, cytokines, and cell-surface proteins. This extension of the capabilities of glycan analytical biotechnologies will help to translate basic science glycobiology to applied glycomedicine and drug discovery.<br>by Vikas Prabhakar.<br>Ph.D.

Le NAD+ apparaît comme un régulateur majeur du fonctionnement mitochondrial. En effet, ce cofacteur régule non seulement l’activité de nombreuses enzymes impliqués dans le métabolisme énergétique (enzymes de la β-oxydation des acides gras, du cycle de Krebs) mais joue également un rôle dans la production d’espèces réactives de l’oxygène (ROS). Le NAD+ est aussi le cofacteur des sirtuines, des enzymes déacétylases régulatrices notamment du métabolisme mitochondrial. De plus, la mitochondrie est l’organite au sein duquel la concentration en NAD+ est la plus élevée (jusqu’à 70% du NAD cellulaire). Le complexe I, qui possède une activité NADH déshydrogénase, pourrait être l’un des régulateurs majeurs du rapport NADH/NAD+ mitochondrial. L’objectif de ce travail de thèse a été d’étudier le rôle du rapport NADH/NAD+ mitochondrial dans le métabolisme énergétique et l’implication du complexe I dans les pathologies mitochondriales. Nous avons mis en évidence qu’une modulation du rapport NADH/NAD+ mitochondrial (augmentation par un activateur pharmacologique ou diminution consécutive à une mutation touchant une sous-unité du complexe I, modifie de manière drastique le métabolisme énergétique notamment en activant ou inhibant la protéine SIRT3, isoforme mitochondriale des sirtuines. Le complexe I semble jouer un rôle majeur dans cette modulation. Le resveratrol, ciblant le complexe I, ainsi que le NMN, un précurseur du NAD+, permettent de restaurer ce rapport et d’améliorer ainsi le métabolisme mitochondrial. Nos résultats suggèrent donc que le rapport NADH/NAD+ pourrait être une cible thérapeutique particulièrement intéressante dans les déficits du complexe I<br>NAD+ appears as a main regulator of the mitochondrial function. Indeed, this compound not only regulates the enzymatic activity of enzymes involved in energetic metabolism (fatty acid oxidation, tricarboxylic acid cycle) but is also involved in ROS production. NAD+ is also the cofactor of sirtuins, deacetylase enzymes, in particular regulating the mitochondrial function. Moreover, mitochondria sequester most of the cellular NAD+ (up to 70 %). The complex I, which possesses an NADH dehydrogenase activity, is thought to be the most important regualtor of the mitochondrial NADH/NAD+ ratio. The work presented here aimed at studying the role of the mitochondrial NADH/NAD+ ratio in mitochondrial metabolism and to test the involvement of the complex I in mitochondrial disorders. We show that a modulation of the mitochondrial NADH/NAD+ ratio (increase by a pharmacological agent or decrease in complex-I mutated fibroplasts) severely affects the mitochondrial energetic function especially by interacting with SIRT3 a mitochondrial sirtuin isoform. The NADH/NAD+ ratio is highly regulated by complex I activity. Resveratrol, which targets the complex I, as well as NMN, a NAD+ precursor, improves the mitochondrial NADH/NAD+ ratio and consequently increases the mitochondrial metabolism. Our results strongly suggest that the mitochondrial NADH/NAD+ ratio could be an interesting therapeutic target especially in complex I- deficient patients

Cocaine abuse is a world-wide public health and social problem without a U.S. Food and Drug Administration (FDA)-approved medication. An ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Recent studies in our lab have led to discovery of the desirable, highly efficient human cocaine hydrolases (hCocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of central nervous system (CNS). Preclinical and clinical data have demonstrated that these hCocHs are safe for use in humans and effective for accelerating cocaine metabolism. However, the actual therapeutic use of a hCocH in cocaine addiction treatment is limited by the short biological half-life (e.g. 8 hours or shorter in rats) of the hCocH. In the investigation described in this thesis, we have demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against cocaine by ~8- and ~2000-fold, respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. In addition, we have identified the first benzoylecgonine-metabolizing enzymes that can hydrolyze benzoylecgonine and accelerate its clearance in rats. The developed LC-MS/MS method has enabled us to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. In development of the long-acting hCocHs, we have designed and discovered a novel hCocH form, catalytic antibody analog, which is an Fc-fused hCocH dimer (hCocH-Fc). The hCocH-Fc has not only a high catalytic efficiency against cocaine, but also a considerably longer biological half-life. A single dose of hCocH-Fc was able to accelerate cocaine metabolism in rats even after 20 days and, thus, block cocaine-induced hyperactivity for a long period of time. In consideration of the general observation that the biological half-life of a protein drug in humans is significantly longer than that in rodents, the hCocH-Fc could allow dosing once every 2-4 weeks, or longer for cocaine addiction treatment in humans.

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes is how they are able to recognize a variety of seemingly unrelated amino acid sequences as cleavage sites. We are investigating the basis for this general substrate recognition in neuropeptidases using thimet oligopeptidase (TOP) as a model. Crystal structures of TOP in complex with a variety of substrates and inhibitors shed light on the mechanisms underlying substrate recognition and pave the way for re-targeting substrate recognition in these enzymes. Nano test tube particles have been proposed as a means of delivering therapeutics such as enzymes. However, the template synthesis method for nano test tube production does not produce therapeutic quantities. In order to take full advantage of re-engineered neuropeptidases a new method for nano test tube synthesis has been developed. We show that a non-destructive template synthesis methodology can be applied to produce nano test tube particles in quantities useful for therapeutic enzyme immobilization.

Insulin-Degrading Enzyme (IDE) is a zinc-metalloprotease responsible for the clearance of insulin in peripheral tissues. Despite decades of speculation that inhibiting endogenous insulin degradation might treat Type-2 Diabetes, the functional relationship between IDE and glucose homeostasis remains unclear. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE’s physiological roles and to determine its potential to serve as a target for the treatment of diabetes. In this thesis I describe the development of the first highly specific IDE in vivo probe, identified from a DNA-templated library of macrocycles, which enabled the first study of the physiological consequences of IDE inhibition. An X-ray structure of the macrocycle bound to IDE reveals that it engages a novel binding pocket away from the catalytic site, which explains its remarkable specificity and its suitability to study IDE in vivo. Treatment of lean and obese mice with this inhibitor revealed that IDE regulates multiple metabolic hormones, including glucagon and amylin, in addition to insulin. Under physiological conditions that mimic a meal, such as oral glucose administration, acute IDE inhibition leads to substantial improvement in glucose tolerance, owing to the potentiation of endogenous insulin and amylin levels over glucagon signaling. These studies demonstrated the feasibility of modulating IDE activity as a therapeutic strategy to treat diabetes and expanded our understanding of the roles of IDE in glucose and hormone regulation. Based on these studies we sought to develop substrate-selective inhibitors that block IDE’s ability to degrade insulin but not its ability to degrade glucagon, which would represent a major step forward towards IDE-targeted therapeutics. The first-generation DNA-templated inhibitor was retailored into a fluorescent anisotropy tool for high-throughput screening of diverse small-molecule libraries. We discovered and characterized a family of IDE inhibitors with sub-micromolar potencies that inherited the remarkable specificity for IDE over other metalloproteases, and selectively obstruct IDE-mediated insulin degradation in a way that accommodates for glucagon cleavage. In conclusion, these findings offer new insights into the biological roles of IDE and establish a novel strategy to selectively potentiate the physiological insulin response in order to improve blood sugar control in Type-2 Diabetes.<br>Chemistry and Chemical Biology

Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.

Thesis (M.D.) - University of Glasgow, 2007.<br>M.D. thesis submitted to the Faculty of Medicine, Dept. of Medicine and Therapeutics, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

The renin-angiotensin system plays a central role in the maintenance of blood pressure. Angiotensin II, the main effector of this system, results from the action of angiotensin-converting enzyme (ACE) on angiotensin I. Angiotensin II, maintains vasomotor tone via its vasoconstrictor action, and also increases salt and water retention by stimulating the release of aldosterone. ACE inhibitors, such as captopril, enalapril and lisinopril, are highly effective in the treatment of hypertension and congestive cardiac failure. Previous studies have suggested that angiotensin converting enzyme (ACE) production may be enhanced during pharmacological inhibition of the enzyme. Little is known, however about the mechanism of this induction. After demonstrating increases in circulating ACE protein in cardiac failure patients receiving the ACE inhibitor captopril, a rat model was used to study this effect. A sensitive enzyme linked immunosorbent assay for rat ACE was developed and a partial cDNA for rat ACE cloned to enable examination of ACE mRNA and protein expression during enzyme inhibition with enalapril. Rat lung ACE mRNA increased by 156% (p<0.05) and ACE protein doubled within 3 hours of administering a single dose of enalapril. Testicular ACE mRNA also increased by 300% (p<0.05) within 2 hours and returned to pretreatment levels by 6 hours. The angiotensin II antagonist saralasin similarly caused a significant (p<0.0001) 800% enhancement of mRNA expression. Aldosterone pretreatment of rats prior to enalapril administration was found to abolish this mRNA induction. These findings indicate that increased ACE expression during inhibition results from reduced levels of angiotensin II with consequent reduced stimulation of the angiotensin 11 receptor and its effects, such as aldosterone release. This suggests that ACE levels are regulated by a negative feedback loop involving the distal components of the renin-angiotensin system, namely angiotensin II and aldosterone. In situ hybridisation and immunohistochemical techniques were employed to localise the site of this inductive response in rat tissue sections. It was found that lung macrophages were markedly induced to produce ACE, as was ACE in seminiferous tubules. ACE induction was also noted in the expected sites of renal tubular epithelium and glomerular tissue. Interestingly, ACE expression was also enhanced in cardiac valves. In these studies it has been conclusively demonstrated that new ACE expression is induced by enzyme inhibitor therapy. A variety of techniques have been developed that will allow futher study of ACE in rat tissues.

The rapid growth of ketamine and amphetamine misuse worldwide has led to the development of methods for the detection and analysis of ketamine and amphetamines in biological specimens. Most methods previously developed in forensic toxicology for the detection of ketamine and amphetamines used GC-MS. The present work developed alternative methods based on LC-MS/MS. Ketamine was chosen as the drug of interest because there are no data currently available on the extent of ketamine abuse in Malaysia even though a large amount of ketamine has been seized by the Malaysian Royal Police, while amphetamines are the most widely abused synthetic drugs in South East Asia including Malaysia. The study addressed some of the challenges facing the forensic toxicologist, such as the need to use new technology (LC-MS/MS) and improve sensitivity and selectivity in forensic toxicology analysis through efficient sample preparation techniques. The general requirements of method validation, including as the parameters linearity, limit of detection (LOD) and Lower Limit of Quantification (LLOQ), recovery, precision and matrix effects were observed. Three main techniques were used in the study: enzyme-linked immunosorbent assay (ELISA), liquid chromatography tandem mass spectrometry (LC-MS/MS) and molecularly imprinted polymer solid phase extraction (MISPE). MISPE is a new extraction technique in forensic toxicology applied to biological samples. Initially work was carried out on the optimization, development and validation of the Neogen® ELISA for screening ketamine and norketamine in urine. The Neogen® ketamine ELISA kit was found to be adequately sensitive and precise for ketamine screening at a cut-off concentration of 25 ng/mL. The ELISA test was shown to be highly specific to ketamine and demonstrated minimal (2.1%) cross-reactivity to its main metabolite norketamine compared to ketamine. Subsequently, an LC–MS/MS confirmation method for ketamine and norketamine in urine samples was developed and validated with application of the method to urine samples from chronic ketamine users in Malaysia. The method demonstrated good linearity, LOD, LOQ, accuracy and precision and had acceptable matrix effects. The efficiency of ELISA as a screening method at cut-off of 25 ng/ml and LC-MS/MS as a confirmation method at 2 ng/ml was evaluated. These methods complemented each other and both ELISA and LC-MS methods were 100% sensitive and specific with no false positive results for ketamine and norketamine in urine samples. The results demonstrated that a combination of these two methods can be reliably used for routine screening and confirmation of ketamine and norketamine in urine specimens. Preliminary data from this study provided information on the concentrations of ketamine and norketamine typically found in urine samples collected from individuals frequenting pubs in Malaysia. The main work in this thesis involved molecularly imprinted polymer materials which were used as sorbents in solid phase extraction (MISPE). Ketamine was used as a model substance for novel in-house synthesised MIPs as no anti-ketamine MIP have previously been reported and because the ketamine structure is suitable for the synthesis of molecularly imprinted polymers. The study was intended to improve the selectivity and sensitivity of the extraction method (MISPE) prior to LC-MS/MS analysis. Evaluation of polymer imprinting was carried out using HPLC-UV. MIP extraction and LC-MS/MS analysis were applied to the determination of ketamine and norketamine in hair samples and compared with a conventional SPE-based method. MISPE extraction was selective and sensitive with fewer matrix effects than the conventional SPE method and could also be applied to norketamine, the principal metabolite of ketamine, due to the group-selective binding nature of the MIP, but not to structurally dissimilar analytes such as PCP and tiletamine. MISPE was superior to conventional SPE for trace detection of ketamine and norketamine in hair, in terms of improved sensitivity, lower limits of detection and reduced matrix effects. In addition, the commercial product Amphetamine SupelMIPTM was evaluated for identification of amphetamines in post mortem blood coupled with LC-MS/MS analysis. This work assessed whether the MIP, sold by the manufacturer for the extraction of amphetamines in urine, could also be used for whole blood. The results demonstrated that the MIP can be used successfully for the determination of amphetamines in post mortem blood. The recoveries of five amphetamines were lower than with a comparable GC-MS method but the LODs and LLOQs of the LC-MS/MS method were better and suitable for detection of low levels amphetamines in post mortem blood. Further optimisation is needed to develop an improved protein precipitation method prior to MISPE. Liquid Chromatography Electro-Spray Ionization Mass Spectrometry (LC–ESI-MS) was used with the MISPE and SPE methods for detection and quantification of ketamine, norketamine and amphetamines in urine, whole blood and hair samples. LC-ESI-MS was found to be easy to use and could detect lower concentrations of drugs and gave reproducible results for all the methods developed in this thesis.